An assay for alkaline phosphatase is described which is based on the hydrolysis of riboflavin phosphates (5'FMN or 4'FMN) to produce riboflavin. This is converted to 5'FMN using riboflavin kinase, and then assayed using the bacterial bioluminescent system from Vibrio harveyi or V. fischeri. The most sensitive assay is obtained using 4'FMN, which can measure less than 20 amol after a 1-hour incubation.